Approach 1: Human Papilloma Virus (HPV) pseudovirions, is a technology developed by my collaborators, Drs Buck, Schiller, and Lowy, at the NCI. The human papilloma virus naturally infects vaginal keratinocytes;using HPV as a vector we can deliver SIV genes directly to the vaginal tract. So with this approach, it may be possible to elicit effector T-cells in the female genital tract. However, a limitation could be that,as the expression of SIV/HIV genes by this vaccine modality wanes with time, repeated immunizations may be required to maintain protective levels of effector T-cells in the vagina In the last year we engineered Human Papiloma virus like particles to express SIV genes. We found that intra-vaginal vaccination with HPV Pseudovirions delivering SIV genes induces both humoral and cell mediated immune responses in macaques. These immune responses were focused in the intraepithelial layers and the lamina propria of the vagina. Upon exposure with SIV we found that vaccine induced immune response quickly expanded in the vaginal tract, vaginal draining lymph nodes within weeks post exposure. This vaccine induced secondary response was however focal and we did not observe an expansion of immune responses in the rectum after intravaginal vaccination or intravaginal SIV challenge, In this current year we wish to test if these focal immune responses induced by HPV Psvs are sufficient to prevent SIV intra-vaginal infection.Vaccine induced effector responses at the portal of entry have been shown to be present in animals resistant to an SIV challenge, however, the only vaccine to show marginal efficacy in humans is a systemically administered canarypox vector ALVAC given in combination with the HIV envelope protein. Thus we wish to test if the combination of vaccine induced effector responses and systemic vaccination can protect from infection. For this dual modality we will prime the immune system with ALVAC SIV and then boost with intravaginal HPV vaccination. We hypothesize that this modality should direct the systemically induced immune responses to the vaginal tract. The rhesus macaque model is the best animal model of HIV infection. It has recently been shown that a single virus establishes most HIV infections and using repeated low dose SIVmac251 mucosal challeges rhesus macaques can also be infected with a single viral variant. W propose using a repeated low dose intra-vaginal challenge with SIVmac251 as our model system to test if our vaccine approaches can protect from SIV infection and/or high levels of SIV viral replication and the loss of CD4+ T cells. Approach 2: Induce effector T-cells in the gastrointestinal tract and will use heat shock gp96-Igbased SIV recombinant vaccines. This is part of a collaborative effort with Dr Podack from the University of Miami and includes the preparation of cellular SIV-gp96-vaccines secreted by 293 cells. Optimal dose-finding for SIV-gp96 vaccines in non-human primates was determined by specific CD8 responses. These cell-based gp96-Ig vaccines, by prolonged in vivo secretion of gp96-Ig-peptide, should imitate viral replication and provide immune stimuli comparable to attenuated viruses. ALVAC is an attenuated canarypox derived vector that cannot replicate productively in mammalian cells. ALVAC HIV-1 vaccine is a candidate HIV-1 vaccine, which is now in Phase III clinical trials and the results of this trail will become available at the end of September 2009. We have designed a study to improve the immunogenicity of an ALVAC SIV vaccine with the goal to test in parallel its efficacy against the high dose or the low dose repeated challenge exposure. In the past, we have tested an ALVAC-SIV vaccine candidate for HIV in an infant macaque model to assess whether this vaccine platform could reduce SIV transmission through breast-feeding. Infant macaques were given multiple immunizations during the first 3 weeks of life with recombinant poxvirus vaccines expressing simian immunodeficiency virus (SIV) structural proteins Gag, Pol, and Env (ALVAC-SIV or modified vaccinia virus Ankara [MVA]-SIV). After repeated daily oral exposure to low doses of virulent SIVmac251 significantly fewer ALVAC-SIV-immunized infants were infected compared with unimmunized infants. Monkeys not infected after oral challenge in infancy were rechallenged at 16 months of age or older by repeated weekly oral SIV exposure;unimmunized animals were infected after fewer SIV exposures than were animals vaccinated with ALVAC-SIV. When infected, ALVAC-SIV -vaccinated animals also had reduced viremia compared with unimmunized animals. These results suggested that immunization of human infants with poxvirus-based HIV vaccine candidates may offer protection against early and late HIV infection and were in contrast with the poor efficacy of these vaccines in adult macaques. It is unclear whether the age or the mode of challenge is responsible for the different degree of protection observed in these two studies. Thus, we are further investigating this approach for the following reasons: 1) ALVAC is an avian vector;therefore, pre-existing immunity is not a factor that could enhance HIV infectivity. This is particularly important in light of the recent results with the Ad5-based HIV vaccine trial (STEP trial) wherein a five fold increase in HIV transmission was observed in individuals (not circumcised)that had high levels of pre-existing immunity to the vector. 2)ALVAC-HIV alone may not be sufficiently immunogenic or protective. 3)We will use this study to test in parallel the degree of protection from a mucosal challenge exposure to high and repeated low doses of SIVmac251. Hopefully, the results of this study compared to the results of the Thailand Trial, will inform on the relevance of the two challenge models in predicting the efficacy of vaccines in humans. We also plan to characterize and compare the viral genotypes that are transmitted early in nave and vaccinated RMs by cloning and sequencing the virus via RT-PCR from the plasma. The scientific goals of this aim are to improve the immunogenicity of an ALVAC-SIV vaccine in a DNA prime /ALVAC-SIV boost and to assess cross-clade protection by testing a SIVmac251-based vaccine against a mucosal challenge with a homologous and heterologous virus, SIVSME660. ALVAC is a plaque-cloned derivative of the Kanapox vaccine strain of the canary poxvirus (CPV).